B-cell receptor (BCR) signaling is a critical pathway in the pathogenesis of several B-cell malignancies, including chronic lymphocytic leukemia (CLL), and can be targeted by inhibitors of BCRassociated kinases, such as Bruton tyrosine kinase (Btk). PCI-32765, a selective, irreversible Btk inhibitor, is a novel, molecularly targeted agent for patients with B-cell malignancies, and is particularly active in patients with CLL. In this study, we analyzed the mechanism of action of PCI-32765 in CLL, using in vitro and in vivo models, and performed correlative studies on specimens from patients receiving therapy with PCI-32765. PCI-32765 significantly inhibited CLL cell survival, DNA synthesis, and migration in response to tissue homing chemokines (CXCL12, CXCL13 IntroductionChronic lymphocytic leukemia (CLL), the most common leukemia in western societies, is characterized by the accumulation of mature, CD5 ϩ CD23 ϩ monoclonal B lymphocytes in the blood, secondary lymphatic tissues, and the bone marrow. 1 Proliferating CLL cells, which account for approximately 0.1% to 1% of the CLL clone, 2 are typically found within microanatomical structures called proliferation centers or pseudofollicles, 3 where CLL cells interact with accessory cells (ie, stromal cells or T cells), thereby receiving survival and growth signals. 4 Such external signals from the leukemia microenvironment can supplement intrinsic oncogenic lesions, thereby promoting maintenance and expansion of the CLL clone. 3,5,6 Among the various external stimuli in the tissue microenvironments, B-cell receptor (BCR) activation and signaling, particularly in lymphatic tissues, 6 is a central pathologic mechanism, even though the precise mechanism of BCR stimulation and the nature of the antigen(s) that activate the BCRs remain obscure. 1,7 The most direct evidence for the importance of BCR signaling in CLL comes from recent comparative gene expression profiling (GEP) data that revealed BCR signaling as the most prominent pathway activated in CLL cells isolated from lymphatic tissues. 6 These GEP changes displayed remarkable similarity to GEP changes of CLL cells cocultured with monocyte-derived nurselike cells (NLC), 8 a system for studying the impact of the lymphatic tissue microenvironment in CLL in vitro. Additional evidence for the importance of BCR signaling in CLL comes from the observation that important CLL risk factors have functional links to the BCRs. The mutation status of the IgV H segments of the BCR distinguishes "mutated" (M-CLL) from "unmutated" CLL (U-CLL), with a low or high risk for disease progression, respectively, each accounting for approximately 50% of the patients. ZAP-70 is predominantly expressed in U-CLL cases, 9 and ZAP-70 expression is associated with enhanced BCR signaling. 10 Furthermore, CLL patients express restricted sets of BCRs, as determined by BCR sequencing. These BCRs have immunoglobulin (Ig) heavy-chain variable (V) gene sequences that are identical or stereotyped in subsets of patients, 11,12 suggesting that these BCR...

IntroductionWith the establishment of more effective treatments for patients with chronic lymphocytic leukemia (CLL) over the past decade, complete remissions are no longer the exception. 1 Despite these major improvements in CLL treatment, we still consider CLL an incurable disease, because patients generally relapse from minimal residual disease (MRD). 2 There is growing evidence suggesting that CLL cells are protected from conventional drugs in tissue microenvironments, such as the bone marrow and secondary lymphoid organs, with facilitation of residual disease that is drug resistant and ultimately paving the way to clonal evolution and relapses. The complex cellular and molecular contexts in the tissues, collectively referred to as the CLL microenvironment, provide signals for the expansion of the CLL clone and for primary drug resistance. This is largely dependent on direct contact between the malignant B cells and stromal cells, 3 and therefore has been designated as cell adhesion-mediated drug resistance. 4 Disrupting cross talk between leukemia cells and their milieu is an attractive novel but yet incompletely tested strategy for treating CLL. Appropriately, there is growing interest in understanding the biology of CLL-stroma cross talk to find ways to eliminate residual CLL cells that are "hiding" in stromal niches within the marrow and the lymphatic tissues.Importantly, once CLL cells are removed from the in vivo microenvironment and placed in suspension cultures without supportive stroma, they undergo spontaneous apoptosis, highlighting the importance of external signals from accessory cells. 5 Previous studies have shown that CLL cell cocultures with different adherent cell types, collectively referred to as stromal cells, induce leukemia cell survival, migration, and drug resistance. These stromal cells include mesenchymal marrow stromal cells (MSCs), 3,6,7 CD68 ϩ nurselike cells derived from monocytes, 7-10 and follicular dendritic cells. 11 Immunohistochemistry showed that in situ, ␣SMA ϩ mesenchymal stromal cells, 12 the in vivo counterpart of MSCs, are a dominant stromal cell population in the CLL microenvironment, which is in contrast to other B-cell lymphomas, particularly high-grade lymphomas, which harbor larger numbers of CD68 ϩ hemangiogenic cells. 12 MSCs regulate normal hematopoiesis by providing attachment sites and secreted or surface-bound growth factors that constitute the marrow microenvironment. 13 During B-cell development in the marrow, programmed cell death regulates B-cell homeostasis by diverting a large fraction of immature B cells into an apoptotic death pathway to eliminate functionless or potentially harmful cells. 14,15 Critical factors for the survival of selected B cells are interactions with MSCs in the marrow microenvironment, [16][17][18] expression of surface immunoglobulin molecules, and expression of apoptosis-regulatory proteins, such as Bcl-2. 19 In patients with CLL, the marrow invariably is infiltrated with CLL B cells, and the For personal use only. on May 7,...

The major concern for anticancer chemotherapeutic agents is the host toxicity. The development of anti-cancer prodrugs targeting the unique biochemical alterations in cancer cells is an attractive approach to achieve therapeutic activity and selectivity. We designed and synthesized a new type of nitrogen mustard prodrug that can be activated by high level of reactive oxygen species (ROS) found in cancer cells to release the active chemotherapy agent. The activation mechanism was determined by NMR analysis. The activity and selectivity of these prodrugs towards ROS was determined by measuring DNA interstrand crosslinks and/or DNA alkylations. These compounds showed 60–90% inhibition toward various cancer cells, while normal lymphocytes were not affected. To the best of our knowledge, this is the first example of H2O2-activated anticancer prodrugs.

Antigenic stimulation through the B-cell antigen receptor (BCR) is considered to promote the expansion of chronic lymphocytic leukemia (CLL) B cells. The spleen tyrosine kinase (Syk), a key component of BCR signaling, can be blocked by R406, a small-molecule Syk inhibitor, that displayed activity in CLL patients in a first clinical trial. In this study, we investigated the effects of BCR stimulation and R406 on CLL cell survival and migration. The prosurvival effects promoted by anti-IgM stimulation and nurselike cells were abrogated by R406. BCR triggering up-regulated adhesion molecules, and increased CLL cell migration toward the chemokines CXCL12 and CXCL13. BCR activation also enhanced CLL cell migration beneath marrow stromal cells. These responses were blocked by R406, which furthermore abrogated BCR-dependent secretion of T-cell chemokines (CCL3 and CCL4) by CLL cells. Finally, R406 inhibited constitutive and BCR-induced activation of Syk, extracellular signal-regulated kinases, and AKT, and blocked BCR-induced calcium mobilization. These findings suggest that BCR activation favors CLL cell homing, retention, and survival in tissue microenvironments. R406 effectively blocks these BCR-dependent responses in CLL cells, providing an explanation for the activity of R406 in patients with CLL.

Purpose Bruton’s tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor pathway and is inhibited by ibrutinib due to covalent binding to the kinase domain. Though ibrutinib results in impressive clinical activity in chronic lymphocytic leukemia (CLL), most patients achieve only partial remission due to residual disease. We performed a pharmacologic profiling of residual circulating CLL cells from patients receiving ibrutinib to identify optimal agents that could induce cell death of these lymphocytes. Experimental design Ex vivo serial samples of CLL cells from patients on ibrutinib were obtained prior and after (weeks 2, 4, and 12) the start of treatment. These cells were incubated with PI3K inhibitors (idelalisib or IPI-145), bendamustine, additional ibrutinib, or BCL-2 antagonists (ABT-737 or ABT-199) and cell death was measured. In vitro investigations complemented ex vivo studies. Immunoblots for BTK signaling pathway and antiapoptotic proteins were performed. Results The BCL-2 antagonists, especially ABT-199, induced high cell death during ex vivo incubations. In concert with the ex vivo data, in vitro combinations also resulted highly cytotoxicity. Serial samples of CLL cells obtained before and 2, 4, 12, or 36 weeks after the start of ibrutinib showed inhibition of BTK activity and sensitivity to ABTs. Among the three BCL-2 family anti-apoptotic proteins that are overexpressed in CLL, levels of MCL-1 and BCL-XL were decreased after ibrutinib while ABT-199 selectively antagonizes BCL-2. Conclusions Our biological and molecular results suggest that ibrutinib and ABT-199 combination should be tested clinically against CLL.

The functional relevance of the B-cell receptor (BCR) and the evolution of protein kinases as therapeutic targets have recently shifted the paradigm for treatment of B-cell malignancies. Inhibition of p110δ with idelalisib has shown clinical activity in CLL. The dynamic interplay of isoforms p110δ and p110γ in leukocytes support the hypothesis that dual blockade may provide a therapeutic benefit. IPI-145, an oral inhibitor of p110δ and p110γ isoforms, sensitizes BCR- stimulated and/or stromal co-cultured primary CLL cells to apoptosis (median 20%, n=57; p<0.0001) including samples with poor prognostic markers, unmutated IgVH (n=28) and prior treatment (n=15) (p<0.0001). IPI-145 potently inhibits the CD40L/IL-2/IL-10 induced proliferation of CLL cells with an IC50 in sub-nanomolar range. A corresponding dose responsive inhibition of pAKTSer473 is observed with an IC50 of 0.36 nM. IPI-145 diminishes the BCR- induced chemokines CCL3 and CCL4 secretion to 17% and 37% respectively. Pre-treatment with 1 μM IPI-145 inhibits the chemotaxis towards CXCL12; reduces pseudoemperipolesis to median 50%, inferring its ability to interfere with homing capabilities of CLL cells. BCR- activated signaling proteins AKTSer473, BADSer112, ERKThr202/Tyr204 and S6Ser235/236 are mitigated by IPI-145. Importantly, for clinical development in hematological malignancies, IPI-145 is selective to CLL B-cells, sparing normal B- and T-lymphocytes.

Resistance to apoptosis in CLL B cells is associated with overexpression of Bcl-2 family antiapoptotic proteins. Their expression is endogenous, but is also induced by signals from the microenvironment resulting in intrinsic and extrinsic drug resistance. Because AT-101 binds to the BH3 motif of all Bcl-2-family antiapoptotic proteins, we hypothesized that this molecule could overcome resistance. AT- IntroductionChronic lymphocytic leukemia (CLL) is characterized by relentless accumulation of mature resting B cells in the peripheral blood, bone marrow and lymphatic tissue. This accumulation is due primarily to defective apoptosis rather than increased proliferation. 1,2 Malignant B cells express high levels of the antiapoptotic protein Bcl-2, either as a result of epigenetic alterations in Bcl-2 gene regulation 3 or because of loss of miR-15a and miR-16-1. 4 Another Bcl-2 family member, Mcl-1, is also overexpressed in CLL B cells 5 ; its expression is associated with survival, chemoresistance, and B-cell receptor signaling. 6 Signals from the microenvironment also amplify antiapoptotic pathways in normal and malignant B cells. 1 Increasing evidence suggests that the interactions between CLL B cells and nonmalignant accessory cells in the bone marrow and lymphatic tissue are pivotal to the maintenance of CLL clone. [7][8][9][10] Accessory cells such as bone marrow stromal cells, 7 monocytederived nurse-like cells, 11,12 and follicular dendritic cells 13 can induce the expression of prosurvival proteins such as 14 In vivo, residual leukemic cells, which may contribute to disease relapse, are harbored in association with stromal cells in the bone marrow/lymphatic tissue. 14 Collectively, this inherent and acquired expression of Bcl-2 family antiapoptotic proteins protects CLL cells from spontaneous and drug-induced apoptosis. Therefore, therapeutic approaches that target these prosurvival proteins within the tissue microenvironment are needed to overcome stromal cell-mediated resistance and improve treatment outcome.Recently, small molecule antagonists are being developed to target prosurvival proteins. These include gossypol, 15 16,19 AT-101 is an enantiomer [R(Ϫ)-gossypol] or -isomer of gossypol that is being tested in phase 1 clinical trials as a single agent for prostate cancer 20 or in combination with rituximab for CLL. 19,21,22 Importantly, AT-101 binds to the BH3 motif of all major antiapoptotic proteins, with high affinity (eg, 230 nM, 570 nM, and 130 nM for Bcl-2, respectively). 22 With these inhibitory properties, we hypothesized that AT-101 could target all major Bcl-2 family antiapoptotic proteins in CLL lymphocytes inducing cell death when grown alone or in presence of microenvironment. To test this postulate, we used a coculture of CLL cells and stromal cells that protect CLL lymphocytes; stromal cell-leukemic cell cocultures are better models for studying the potential of in vivo interactions to perpetuate CLL B-cell survival. MethodsAll patients provided written informed consent in accordance wi...

Purpose Ibrutinib inhibits Bruton tyrosine kinase (BTK) by irreversibly binding to the Cys-481 residue in the enzyme. However, ibrutinib also inhibits several other enzymes that contain cysteine residues homologous to Cys-481 in BTK. Patients with relapsed/refractory or previously untreated chronic lymphocytic leukemia (CLL) demonstrate a high overall response rate to ibrutinib with prolonged survival. Acalabrutinib, a selective BTK inhibitor developed to minimize off-target activity, has shown promising overall response rates in patients with relapsed/refractory CLL. A head-to-head comparison of ibrutinib and acalabrutinib in CLL cell cultures and healthy T cells is needed to understand preclinical biologic and molecular effects. Experimental Design Using samples from patients with CLL, we compared the effects of both BTK inhibitors on biologic activity, chemokine production, cell migration, BTK phosphorylation, and downstream signaling in primary CLL lymphocytes and on normal T-cell signaling to determine effects on other kinases. Results Both BTK inhibitors induced modest cell death accompanied by cleavage of PARP and caspase 3. Production of CCL3 and CCL4 chemokines and pseudoemperipolesis were inhibited by both drugs to a similar degree. These drugs also showed similar inhibitory effects on phosphorylation of BTK and downstream S6 and ERK kinases. By contrast, off-target effects on SRC-family kinases were more pronounced with ibrutinib than acalabrutinib in healthy T lymphocytes. Conclusion Both BTK inhibitors show similar biological and molecular profile in primary CLL cells but appear different on their effect on normal T-cells.